Antigen Processing and Presentation Protocols (Methods in by Solheim J.C.

By Solheim J.C.

Well-recognized and leading edge experimentalists element their state of the art equipment for learning the antigen processing and presentation. Drawing on services from biochemistry, mobilephone biology, and immunology, they describe step by step tools designed to question how MHC-binding peptides are generated, to check how peptides are brought to MHC molecules, to research MHC peptide-binding styles, and to assay the T-cell reaction to the MHC/peptide complicated. Emphasis is given these technical steps serious for experimental good fortune which are usually passed over from tools released within the fundamental literature. Eminently available and cutting-edge, Antigen Processing and Presentation Protocols presents either new and skilled investigators with hugely sensible instruments that might expand the questions that may be requested, and successfully be responded, touching on antigen processing/presentation.

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1. 35S-L-methionine (Met) (10 mCi/mL). 2. 2% bovine serum albumin (BSA) (PBS/BSA). 3. Methionine (Met) starving medium: Met-free, serum-free medium (RPMI or Dulbecco’s modified Eagle’s medium [DMEM]), containing 20mM HEPES. 4. PBS containing 10 mM L-Met (PBS–Met). 5. 5% fetal calf serum (FCS) and L-Met (I/Met). 6. rVV expressing the chimeric protein UbRNP. 7. 5% (v/v) 2-mercaptoethanol. 8. Protease inhibitor cocktail. Boehringer Mannheim’s (Indianapolis, IN) Complete inhibitors work well for this purpose.

Use 270 µL for washes. 8. If the first Ab is unlabeled, wash cells in PBS–BSA, and incubate with a FITC-labeled Ab, specific for the class I Ab used, and incubate for 1 h. 9. , whether using labeled or unlabeled anticlass I Ab, wash the cells with PBS–BSA and finally resuspend in 400 µL PBS–BSA containing 10 µg/mL ethidium homodimer (see Note 8). Using a multichannel pipetor transfer cells to 1 mL conical tubes arrayed in a 96-well format, and keep on ice. 10. Analyze the cells in a flow cytometer, gating out dead cells as identified by positive staining for ethidium homodimer.

1. Pulsing Fixed Cells with Ag Cells lightly fixed with paraformaldehyde still express peptide-receptive MHC-I molecules at the cell surface, and are able to present peptide–MHC-I complexes to T-cells. Therefore, fixed cells represent an ideal way to screen for peptide contamination in the absence of cellular processes. In addition, over prolonged periods, this assay can also reveal the action of serum proteases in the generation of MHC-I binding peptides. For protein Ags and inactivated infective agents (inactivated viruses, and so on): 1.

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